An antibody targeting CD47 and its application

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to an antibody targeting CD47 and its application. The anti-human CD47 antibody provided by the present invention can effectively bind to recombinant CD47 protein, block the binding of CD47 to its biological ligand SIRPa, and effectively identify and bind to CD47 positive tumor cells. At the same time, the CD47 antibody does not cause significant hemagglutination reaction and red blood cell binding of human red blood cells at high concentrations.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to an antibody targeting CD47 and an application thereof. Background Art

[0002] To date, immunotherapy is considered to be the most promising systemic tumor treatment method compared with traditional anti-cancer treatment strategies, and plays an indispensable role in improving treatment efficacy. Emerging cancer immunotherapies include cancer vaccines, CAR-T cell therapy, cytokine therapy, immune checkpoint inhibitors, and tumor-targeted monoclonal antibodies. Among them, monoclonal antibodies have become a key and effective treatment method in cancer treatment due to their ability to specifically target molecules.

[0003] Yeast surface display technology is a set of general technologies used in the fields of antibody discovery, target discovery and protein engineering. As one of the technical means for developing therapeutic antibodies in vitro, it has achieved progress from prokaryotic expression systems to eukaryotic expression systems. In antibody engineering, the characteristics of the yeast eukaryotic expression system are used to express antibody library proteins on the surface of yeast cells. Magnetic beads and flow cytometry sorting technology are used to screen based on antibody affinity and display level to obtain high-affinity or high-stability antibody sequences. According to the design, antibodies with a variety of skeleton forms can be obtained, such as single-chain variable region scFv, antibody binding region fragment Fab, full-length IgG, camelid single-domain antibody variable region VHH. At the same time, the eukaryotic system can be used to post-translationally modify antibodies, and the antibody characteristics are closer to antibodies produced by mammalian cells.

[0004] Leukocyte differentiation antigen CD47 (cluster of differentiation 47), also known as integrin-associated protein (IAP), is a glycosylated transmembrane protein widely expressed on the surface of a variety of cells. The molecular weight of CD47 is 45-55kDa. Its structure includes an extracellular variable region that interacts with the corresponding ligand, a transmembrane region formed by a highly hydrophobic transmembrane segment, and a hydrophilic carboxyl-terminal intracellular region. It belongs to the immunoglobulin superfamily. CD47 and its ligands not only regulate immune responses, but also mediate various pathological and physiological processes, such as neutrophil chemotaxis and nervous system development, and play a regulatory role in immune tolerance and T cell activation. Signal regulatory protein α (SIRPα) is its specific ligand, which is mainly expressed on the surface of phagocytic cells such as macrophages and dendritic cells. The interaction between CD47 / SIRPα can transmit inhibitory signals and inhibit the phagocytosis of related cells by macrophages.

[0005] A large number of studies have shown that CD47 is overexpressed in different types of tumors, and high CD47 expression levels are associated with worsening treatment response and prognosis of cancer. Tumor cells transmit "don't eat me" signals to macrophages through CD47 / SIRPα interaction, which leads to the escape of tumor cells' inherent immunity and induces further occurrence and development of tumors. Therefore, blocking the inhibitory signals mediated by the CD47 / SIRPα axis and restoring the phagocytic effect of macrophages on tumor cells provide new ideas and means for tumor immunotherapy.

[0006] However, due to the widespread expression of CD47, potential problems with anti-CD47 antibodies as anticancer drugs may result in off-target effects, such as anemia. CD47 is a key regulator of red blood cell renewal, and CD47-targeted antibodies usually have a high targeting affinity with red blood cells, which may accelerate red blood cell clearance and lead to hemolytic anemia. For example, CD47-targeted agents (i.e., Hu5F9-G4 and TTI-621) can cause acute anemia and thrombocytopenia in humans. Therefore, obtaining anti-CD47 monoclonal antibodies with high activity and low toxicity and side effects is of great significance for tumor immunotherapy. Summary of the invention

[0007] Purpose of the invention: The present invention provides a new antibody which not only has good affinity with CD47 but also has weak red blood cell agglutination.

[0008] The present invention provides a novel CD47 antibody or an antigen-binding fragment thereof and a method for preparing the same. The provided CD47 antibody can recognize and specifically bind to CD47, and can be used to prepare drugs for treating diseases related to abnormal or excessive CD47 expression without causing significant hemagglutination reaction; or can be used to prepare products for detecting CD47 expression by Western blot, ELISA, or flow cytometry.

[0009] An anti-human CD47 antibody or an antigen-binding fragment thereof, characterized in that the antibody or antigen-binding fragment specifically binds to human CD47 and comprises the following VH CDR1, VH CDR2 and VH CDR3 of the heavy chain variable region and VL CDR1, VL CDR2 and VL CDR of the light chain variable region, wherein:

[0010] VH CDR1 comprises any one of the amino acid sequences shown in SEQ ID NOs: 8-10;

[0011] VH CDR2 comprises any one of the amino acid sequences shown in SEQ ID NOs: 11-13;

[0012] VH CDR3 comprises any one of the amino acid sequences shown in SEQ ID NOs: 14-16;

[0013] VL CDR1 comprises any one of the amino acid sequences shown in SEQ ID NOs: 17-19;

[0014] VL CDR2 comprises any one of the amino acid sequences shown in SEQ ID NOs: 20-22;

[0015] VL CDR3 comprises any one of the amino acid sequences shown in SEQ ID NOs: 23-26.

[0016] An anti-human CD47 antibody or an antigen-binding fragment thereof, characterized in that:

[0017]

[0018] The anti-human CD47 antibody or antigen-binding fragment thereof is characterized in that:

[0019]

[0020] A genetically engineered antibody, characterized in that the heavy chain and light chain amino acid sequences it contains are consistent with the amino acid sequences described in claim 3; the genetically engineered antibody includes: human-mouse chimeric antibodies; or humanized antibodies; or functional fragments of antibodies Fab; or single-chain antibodies; or functional fragments of antibodies VH-L fused with heavy chain variable regions and complete light chains; or functional fragments of antibodies formed by arrangement, concatenation or combination of one or more CDRs of heavy and light chains; or functional fusion proteins of antibody-like nature obtained by connecting, splicing or fusing the above antibodies and antibody functional fragments with various other proteins or polypeptides.

[0021] The anti-CD47 antibody or its antigen-binding fragment or the genetically engineered antibody is characterized in that the antibody has a constant region, the heavy chain constant region is selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is a κ or λ chain. The nucleotide sequence encoding the anti-CD47 antibody or its antigen-binding fragment or the genetically engineered antibody.

[0022] An expression vector comprises the nucleotide sequence encoding the above-mentioned gene.

[0023] The use of the anti-CD47 antibody or its antigen binding fragment or the genetically engineered antibody in the preparation of anti-tumor drugs.

[0024] Beneficial Effects

[0025] The present invention provides a series of full-length antibodies or single-chain antibodies, which have good anti-CD47 effect and weak or no red blood cell aggregation effect, and have good application prospects in clinic. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Indirect ELISA is used to detect the binding of CD47 antibody to recombinant human CD47 antigen. Figure A shows the EC50 fitting curves of antibodies D0604-G4 and D0643-G4; Figure B shows the EC50 fitting curves of D2510-G4, D3705-G4 and the positive control antibody Hu5F9-G4.

[0027] Figure 2 Flow cytometry was used to determine the ability of CD47 antibodies to bind to tumor cells expressing CD47.

[0028] Figure 3 The results are shown in Figure 2. CD47 antibody induced hemagglutination of red blood cells at different concentrations.

[0029] Figure 4 The binding activity of CD47 antibody to red blood cells was measured by flow cytometry. DETAILED DESCRIPTION

[0030] The present invention provides antibodies that specifically bind to CD47. In some embodiments, the antibodies of the present invention can specifically bind to human CD47. Herein, these antibodies are collectively referred to as CD47 antibodies.

[0031] The term "monoclonal antibody" (mAb) refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies contained in the population are identical except for a few naturally occurring mutations that may be present. It contains only one molecular species of antibody molecules composed of a unique light chain gene product and a unique heavy chain gene product. Specifically, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all molecules of the population. mAbs contain an antigen binding site that is capable of immunoreacting with a specific epitope of the antigen.

[0032] The term "single-chain antibody" (scFv) refers to an antibody composed of the heavy chain variable region (VH) and the light chain variable region (VL) of an antibody connected by a short peptide linker of 15-20 amino acids.

[0033] The term "complementarity determining region" or "CDR" refers to the hypervariable regions of the heavy and light chains of immunoglobulins. There are three heavy chain CDRs and three light chain CDRs. Here, depending on the circumstances, the terms "CDR" and "CDRs" are used to refer to the region containing one or more or even all of the major amino acid residues that contribute to the binding affinity of an antibody or antigen-binding fragment thereof to its recognized antigen or epitope. In another specific embodiment, the CDR region or CDR refers to the hypervariable regions of the heavy and light chains of immunoglobulins as defined by IMGT.

[0034] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, cell culture, recombinant DNA technology, and related techniques in the art.

[0035] Unless otherwise specified, the reagents required for the following embodiments can all be obtained from commercial sources (commercially available).

[0036] Example 1: Immunization of mice

[0037] 1) Select healthy Balb / c female mice aged 6-8 weeks and use His-tagged hCD47 recombinant protein for five immunizations. For the first immunization, 100ug of antigen protein was mixed and emulsified with Freund's complete adjuvant at a ratio of 1:1, and immunization was completed by subcutaneous multi-point injection. Thereafter, four booster immunizations were performed, with an interval of 14 days between each booster immunization. The amount of antigen was halved and mixed and emulsified with Freund's incomplete adjuvant at a ratio of 1:1, and immunization was completed by intraperitoneal injection. After each booster immunization, part of the mouse tail was cut off, the mouse tail vein blood was collected and the serum was separated, and the mouse serum titer was determined by ELISA.

[0038] 2) Dilute the hFc-tagged hCD47 recombinant protein to 1 μg / ml with antigen coating solution, add 100 μl / well to the UV-activated enzyme label strip, set up duplicate wells, and set up a PBS group as a negative control, and coat overnight at 4°C. Aspirate the coating solution, add PBST to wash each well, each time for 5 minutes, wash 6 times; add 5% skim milk blocking solution to each well, block at 37°C for 2 hours; aspirate the blocking solution, add PBST to wash each well, each time for 5 minutes, wash 6 times; add mouse serum diluted with PBS in different proportions to each well, incubate at 37°C for 2 hours; aspirate the mouse serum, add PBST to wash each well, each time for 5 minutes, wash 6 times; add diluted HRP-goat anti-mouse IgG antibody to each well, incubate at 37°C for 1.5 hours; aspirate the secondary antibody, add PBST to wash each well, each time for 5 minutes, wash 6 times; add freshly prepared TMB color development solution to each well, color at 37°C in the dark for 15-30 minutes; add stop solution to each well to stop the color development reaction, and measure the absorbance of each well at a wavelength of 450nm to determine the serum titer after mouse immunization.

[0039] Example 2: Screening and preparation of CD47 antibodies

[0040] 1. Extraction of total RNA from mouse spleen

[0041] 1) According to the results of ELISA, the mice with the highest serum titer after booster immunization were selected, and the mice were killed by cervical dislocation and spleen was isolated to prepare a single cell suspension of spleen tissue. After the red blood cells were removed by red blood cell lysis buffer, TRizol reagent was added and the cells were repeatedly blown to fully lyse the cells.

[0042] 2) Add 1 / 5 volume of chloroform to the above lysate, let stand at room temperature for 2-3 minutes; centrifuge at 4°C, 12000g for 10-15 minutes. Carefully pipette the upper aqueous phase into a new centrifuge tube, add 1 / 2 volume of isopropanol, mix thoroughly, and let stand at room temperature for 10 minutes; centrifuge at 4°C, 12000g for 10 minutes. Discard the supernatant, add an equal volume of 75% ethanol to wash the precipitate, and let it air dry at room temperature for 5-10 minutes. Add 50μl of RNase-free water to dissolve the RNA.

[0043] 2.RT-PCR

[0044] According to the variable region nucleic acid sequence of mouse antibody, specific primers were designed, and the mouse heavy chain variable region (VH) and light chain variable region (VL) genes were amplified by PCR using the first chain of cDNA as a template. The DNA fragment of mouse single-chain antibody (scFv) was obtained by overlap PCR, and the scFv fragment was purified by gel recovery after 1% agarose gel electrophoresis.

[0045] 3. Construction of CD47 Yeast Antibody Library and Antibody Screening

[0046] 1) Library construction: The yeast vector pYD1 was digested with EcoRI and XhoI restriction endonucleases at 37°C overnight, and the linearized vector was recovered after 1% agarose gel electrophoresis. It was co-transformed with the purified scFv gene fragment into the cerevisiae strain EBY100 by electroporation and cultured at 30°C, 220 rpm overnight to display the single-chain antibody on the yeast cell surface. The corresponding single-chain antibody library was constructed and named DEM139.

[0047] 2) Magnetic bead sorting: Determine the OD of the yeast solution after electroporation 600 , inoculated in the induction medium at an appropriate density, and induced and cultured overnight at 20°C and 220rpm; the induced yeast was taken, washed with 1% PBSA, and the hFc-tagged CD47 antigen protein labeled with biotin was added, incubated at room temperature for 20min, and the yeast was washed with pre-cooled 1% PBSA, placed on a magnetic rack, and sorted using streptavidin magnetic beads, and the yeast was collected with amplification medium, and cultured overnight at 30°C and 220rpm.

[0048] 3) Flow cytometry: Determine the OD of yeast culture after magnetic bead separation 600 , inoculate in the induction medium at an appropriate density, culture overnight at 20°C and 220rpm, and measure the OD of the bacterial solution 600, draw an appropriate amount of yeast, wash with PBS, centrifuge at 3000rpm for 5min, remove the supernatant, resuspend the yeast with biotinylated hFc-tagged CD47 antigen protein, incubate at room temperature for 20min, wash the yeast with pre-cooled PBS, centrifuge at 3000rpm for 5min, remove the supernatant, add diluted APC-tagged streptavidin and Alexa Mouse anti-V5 tag antibody labeled with 488 was incubated at 4°C for 30 min, washed with pre-cooled PBS, centrifuged at 3000 rpm for 5 min, supernatant removed, cells resuspended in PBS, gated according to fluorescence intensity, APC and 488 double-positive yeast cells were sorted into amplification medium, cultured overnight at 30°C, 220 rpm, and induced again for a second round of flow sorting.

[0049] 4) Library sequencing: After two rounds of flow sorting, yeast amplification culture was taken, yeast plasmids were extracted, and library sequencing was performed. A total of 50 single clones with better growth status were selected for sequencing.

[0050] 4. Preparation of CD47 Antibody

[0051] 1) Construction of eukaryotic expression vector: According to the sequencing results of the library, four single-chain antibody sequences with different CDR sequences were selected, primers were designed, and the antibody light chain variable region and the human Igκ chain constant region were spliced ​​by overlap PCR, and the antibody heavy chain variable region and the human IgG4 constant region were spliced, thereby converting each single-chain antibody into a full-length antibody. The antibody light and heavy chain genes were spliced ​​into a mammalian expression vector by homologous recombination to obtain the corresponding CD47 full-length antibody eukaryotic expression vector.

[0052] 2) The combinations of VH and VL of the four IgG4 full-length antibodies are shown in Table 1; the C-terminus of VH is connected to the CH of a sequence such as SEQ ID NO: 27 to form the heavy chain of the antibody; the C-terminus of VL is connected to the CL of a sequence such as SEQ ID NO: 28 to form the light chain of the antibody; the heavy chain variable region of the antibody is represented by "D" + the antibody heavy chain number + "-VH", such as D06-VH; the light chain variable region of the antibody is represented by "D" + the antibody light chain number + "-VL", such as D04-VL; the IgG4 full-length antibody is represented by "D" + the antibody heavy chain number + the antibody light chain number + "-G4", such as D0604-G4 (the antibody heavy chain consists of D06-VH (SEQ ID NO: 1) and the CH of a sequence such as SEQ ID NO: 27, and the antibody light chain consists of D04-VL (SEQ ID NO: 4) and the sequence such as SEQ ID NO: 28), and the four full-length antibodies screened were abbreviated as D0604-G4, D0643-G4, D2510-G4 and D3705-G4.

[0053] 3) The recombinant plasmids encoding the light and heavy chains of the CD47 antibody and the positive control antibody Hu5F9-G4 (the sequence of Hu5F9-G4 has been disclosed in U.S. Patent US20150183874A1) were transfected into HEK293 cells at a ratio of 2:1. After culturing for 5 days, the supernatant was collected by centrifugation at 4°C, and the CD47 antibody was purified by protein A column. The antigen molecular weight and purity and antibody type were detected by SDS-PAGE, SEC-HPLC and Western blot, respectively.

[0054] The results showed that the molecular weight of the antibody was 150-160kd and the type was IgG.

[0055] Table 1: Full-length antibody formed by combining the heavy chain variable region and light chain variable region of CD47 antibody

[0056]

[0057] Table 2: CDR regions of antibodies

[0058]

[0059] Example 3: Determination of the binding ability and affinity of CD47 antibodies to hCD47 molecules

[0060] Use ELISA to detect the binding of CD47 antibody to recombinant human CD47 antigen. Dilute the mFc-tagged hCD47 recombinant protein to 1μg / ml with antigen coating solution, add 100μl / well to the UV-activated enzyme label strip, set up duplicate wells, and set up the PBS group as a negative control, and coat overnight at 4°C. Wash 3 times with PBST, add 5% skim milk, and block at 37°C for 2h. Wash 3 times with PBST, add gradient dilutions of CD47 antibody and control antibody Hu5F9 (highest concentration 200nM, a total of 8 concentration gradients) and incubate at 37°C for 2h. Wash 3 times with PBST, add HRP-labeled goat anti-human IgG secondary antibody, incubate at 37°C for 1h, wash 6 times with PBST, add TMB colorimetric solution, incubate in the dark for 15min, and terminate the colorimetric reaction. Read the absorbance at 450nm on the microplate reader, calculate the EC50, and the results are as follows Figure 1 As shown in Table 3, each CD47 antibody can effectively bind to hCD47 protein at the protein level.

[0061] Table 3: Affinity determination results of CD47 antibody and hCD47 molecule

[0062] Antibody ID EC50(nM) D0604-G4 0.3457 D0643-G4 0.0853 D3705-G4 0.2218 D2510-G4 2.6910 Hu5F9-G4 0.0570 .

[0063] Example 4: Determination of the effect of CD47 antibody blocking the binding of CD47 and SIRPα

[0064] Dilute the hFc-tagged hCD47 recombinant protein to 1 μg / ml with antigen coating solution, add 100 μl / well to the UV-activated enzyme label strip, set up duplicate wells, and set up the PBS group as a negative control, and coat at 4°C overnight. Wash with PBST 3 times, add 5% skim milk, and block at 37°C for 2h. Wash with PBST 3 times, add gradient dilutions of CD47 antibody and control antibody Hu5F9 (maximum concentration 300nM, a total of 10 concentration gradients) and biotin-labeled mFc-tagged SIRPα recombinant protein with a final concentration of 2 μg / ml, and incubate at 37°C for 1h. Wash with PBST 3 times, add HRP-labeled streptavidin, incubate at 37°C for 1h, wash 6 times with PBST, add TMB colorimetric solution, incubate in the dark for 15min, and terminate the colorimetric reaction. The absorbance at 450 nm was read on an ELISA reader, and the IC50 of each antibody was calculated. The results are shown in Table 4. The CD47 antibody can effectively block the interaction between CD47 and SIRPα.

[0065] Table 4: Results of the assay of CD47 antibody blocking the binding of CD47 to SIRPα

[0066] Antibody ID IC50(nM) D0604-G4 8.594 D0643-G4 5.196 D3705-G4 5.025 D2510-G4 41.090 Hu5F9-G4 0.456

[0067] Example 5: Determination of the binding ability of CD47 antibodies to tumor cells expressing CD47

[0068] Jurkat cells with high expression of CD47 were cultured in 1640 medium containing 10% FBS at 37°C and 5% CO2. When the cells were growing well, they were counted and 2×10 5 Cells were centrifuged at 1500 rpm at 4°C for 5 min, and the medium was discarded; cells were resuspended with 100 μl PBS containing 2% BSA and blocked at 4°C for 20 min; centrifuged at 1500 rpm at 4°C for 5 min, and the supernatant was discarded; cells were resuspended with 100 μl 100 nM CD47 antibody and incubated at 4°C for 1 h; centrifuged at 1500 rpm at 4°C for 5 min, and the primary antibody was discarded; cells were resuspended and washed with 500 μl PBS, and centrifuged at 1500 rpm at 4°C for 5 min, and the supernatant was discarded; 100 μl diluted Alexa Resuspend the cells with 488 labeled goat anti-human IgG antibody, incubate at 4℃ for 30min, centrifuge at 1500rpm, 4℃ for 5min, and discard the secondary antibody; resuspend and wash the cells with 500μl PBS, centrifuge at 1500rpm, 4℃ for 5min, discard the supernatant, repeat once; add 250ul PBS to resuspend the cells, filter through a 200-mesh cell screen, and use a flow cytometer for detection. The results are as follows Figure 2As shown, the CD47 antibody of the present invention can effectively bind to tumor cells with high CD47 expression and has good tumor cell targeting ability.

[0069] Example 6: Detection of the effect of CD47 antibody on human red blood cell (RBC) agglutination

[0070] Fresh blood was collected from healthy donors, lymphocytes were separated using lymphocyte separation solution, 500 μl of red blood cells were aspirated after centrifugation, 3 ml of PBS was added to wash the cells, and the cells were centrifuged at 2000 rpm at room temperature for 5 min, for a total of three washes; the red blood cells were resuspended and diluted to a 6% red blood cell suspension with PBS, and added to a 96-well plate for red blood cell plating, with 50 μl of cell suspension in each well; the highest final incubation concentration of each CD47 antibody was set to 300 nM, and it was diluted 2-fold with PBS continuously, for a total of 12 concentration gradients; 50 μl of CD47 antibody of each concentration gradient was added to each well, incubated at 37°C for 4 h, and the aggregation of red blood cells in each well was observed. The results are as follows Figure 3 As shown in the figure, among the CD47 antibodies tested, antibody D3705-G4 (300-9.375 nM) and HU5F9-G4 induced erythrocyte agglutination at high concentrations (300-1.172 nM). Antibodies D0604-G4 and D0643-G4 had slight erythrocyte agglutination at high concentrations (300-150 nM), while D2510-G4 did not show obvious erythrocyte agglutination even at the highest concentration.

[0071] Example 7: Detection of the binding ability of CD47 antibody to human red blood cells (RBC)

[0072] 20 μl of 6% red blood cell suspension was drawn from each tube, and the cells were resuspended in PBS containing 2% BSA, blocked at 4°C for 20 min, centrifuged at 1500 rpm, 4°C for 5 min, and the supernatant was discarded; the highest final incubation concentration of each CD47 antibody was set to 200 nM, and PBS was used for continuous 2-fold dilution, with a total of 10 concentration gradients, 100 μl of red blood cells were resuspended in each tube, and incubated at 4°C for 1 h; the cells were resuspended and washed once with 500 μl PBS; 100 μl of diluted Alexa Resuspend the cells with goat anti-human IgG antibody labeled with 488, incubate at 4℃ for 30min, centrifuge at 1500rpm, 4℃ for 5min, and discard the secondary antibody; resuspend and wash the cells twice with 500μl PBS, add 1ml PBS to resuspend the cells, filter through a 200-mesh cell screen, and then use a flow cytometer for detection. The results are as follows Figure 4As shown, among the tested CD47 antibodies, antibodies D0604-G4, D2510-G4 and Hu5F9-G4 all showed the ability to bind to human red blood cells to varying degrees, and this ability was dose-dependent. In particular, D0604-G4 and D2510-G4 showed lower binding to red blood cells, suggesting that they may have lower erythrocyte toxicity than Hu5F9-G4.

Claims

1. An anti-human CD47 antibody or an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment specifically binds to human CD47 and comprises a heavy chain variable region and a light chain variable region; wherein the heavy chain variable region consists of VH CDR1, VH CDR2 and VH CDR3; and the light chain variable region consists of VL CDR1, VL CDR2 and VL CDR3. in: D0604-G4: VH CDR1, VH CDR2 and VH CDR3 are the amino acid sequences shown in SEQ ID NOs: 8, 11 and 14, respectively; VL CDR1, VL CDR2 and VL CDR3 are the amino acid sequences shown in SEQ ID NOs: 18, 21 and 23, respectively; D0643-G4: VH CDR1, VH CDR2 and VH CDR3 have the amino acid sequences shown in SEQ ID NOs: 8, 11 and 14, respectively; VLCDR1, VL CDR2 and VL CDR3 have the amino acid sequences shown in SEQ ID NOs: 17, 20 and 26, respectively; D2510-G4: VH CDR1, VH CDR2 and VH CDR3 are the amino acid sequences shown in SEQ ID NOs: 9, 12 and 15, respectively; VLCDR1, VL CDR2 and VL CDR3 are the amino acid sequences shown in SEQ ID NOs: 17, 20 and 25, respectively; D3705-G4: VH CDR1, VH CDR2 and VH CDR3 have the amino acid sequences shown in SEQ ID NOs: 10, 13 and 16, respectively; VLCDR1, VL CDR2 and VL CDR3 have the amino acid sequences shown in SEQ ID NOs: 19, 22 and 24, respectively.

2. An anti-human CD47 antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The heavy chain variable region of D0604-G4 has the amino acid sequence shown in SEQ ID NO: 1; the light chain variable region has the amino acid sequence shown in SEQ ID NO: 4; D0643-G4: the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 1; the light chain variable region has the amino acid sequence shown in SEQ ID NO: 7; D2510-G4: the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 2; the light chain variable region has the amino acid sequence shown in SEQ ID NO: 6; D3705-G4: The heavy chain variable region has the amino acid sequence shown in SEQ ID NO:3; the light chain variable region has the amino acid sequence shown in SEQ ID NO:

5.

3. The anti-CD47 antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The antibody has a constant region, wherein the heavy chain constant region is selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; and the light chain constant region is a κ or λ chain.

4. A genetically engineered antibody, characterized in that: It contains the anti-human CD47 antibody or its antigen binding fragment as claimed in claim 2; the genetically engineered antibody includes: human-mouse chimeric antibody; or humanized antibody; or functional fragment Fab of antibody; or single-chain antibody.

5. A nucleotide sequence encoding the anti-CD47 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 or the genetically engineered antibody according to claim 4. An expression vector comprising the nucleotide sequence encoding the gene of claim 5 .

7. Use of the anti-CD47 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 or the genetically engineered antibody according to claim 4 in the preparation of an anti-tumor drug, characterized in that: The tumor is a tumor with high expression of CD47, and the tumor is selected from leukemia, lymphoma, lung cancer, breast cancer, colorectal cancer, melanoma or ovarian cancer.

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